Impaired activation of glycogen synthase in insulin-resistant subjects is associated with both chronically lower activity of protein phosphatase-1 (PP1) and a smaller magnitude of activation of PP1 in response to insulin compared to that seen in insulin-sensitive subjects. Although PP1 activity is low in muscle of resistant subjects, the concentration of catalytic subunit of PP-1alpha is higher than in sensitive subjects. The predicted amino acid sequence of the catalytic domain of a 420 bp PP- 1alpha PCR product was examined in insulin sensitive and resistant subjects, and found to be identical. PP1-alpha RNA concentration and size in skeletal muscle (1.6 kb) were also the same in fasting subjects of different insulin sensitivities. The concentration of RNA corresponding to PP1-alpha in human skeletal muscle decreased by 50% after 30 min of a high dose infusion of insulin in vivo. The concentration returned to basal levels by 120 min. In contrast, PP1-alpha RNA increases slightly in response to insulin in insulin-resistant subjects. The cDNA sequence for another PP-1 catalytic subunit isoform from human muscle was found to be 93% similar in coding sequence to PP-1 gamma-1, previously described in rat liver. This isoform was detected by Northern blot in human heart, brain, placenta, lung, liver, skeletal muscle kidney and pancreas. The specific PP-1 gamma-1 sequence was localized to human chromosome 12.